This Insulin (INS) BioAssay™ ELISA Kit is a sandwich ELISA for the in vitro quantitative determination of INS concentrations in rat serum, plasma and other biological fluids.||Sensitivity:|0.19ng/ml||Detection Range:|0.31-20ng/ml||Precision:|Intra-Assay %CV: <10%|Inter-Assay %CV: <10%||Specificity:|Recognizes rat INS. No significant cross-reactivity or interference between rat INS and analogues was observed.||Test Principle:|The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to rat INS. Standards or samples are added to the micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for rat INS and Avidin-Horseradish Peroxidase (HRP) conjugate are added successively to each micro plate well and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain rat INS, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by the addition of stop solution and the color turns yellow. The optical density (O.D.) is measured spectrophotometrically at a wavelength of 450nm ± 2nm. The O.D. value is proportional to the concentration of rat INS. You can calculate the concentration of rat INS in the samples by comparing the O.D. of the samples to the standard curve.||Kit Components:|Microtiter Strips, 1x96 wells, Pre-coated; ready to use|Standard, 2x1vial |Standard Diluent, 1x20ml|Biotin Detection, 100X, 1x120ul |HRP Conjugate, 100X, 1x120ul |Biotin Detection Diluent, 1x14ml|HRP Diluent, 1x14ml|Substrate, 1x10ml|Stop Solution, 1x10ml|Wash Buffer, 25X, 1x30ml ||Storage and Stability:|Store Microtiter Strips, Standard, Detection Reagents and Positive Control at -20°C. After reconstitution of Positive Control, store at 4°C and use within 5 days. Store all the other components at 4°C. Unused kit is stable for 6 months after receipt. Once kit components are opened, it is highly recommended to use remaining reagents within 1 month provided this is within the expiration date of the kit. For maximum recovery of product, centrifuge the original vials after thawing and prior to removing the cap.||Assay Procedure Summary:|1. Prepare all reagents, samples and standards.|2. Add 100ul standard or sample to each well. Incubate 90 minutes at 37°C.|3. Aspirate and add 100ul prepared Biotin Working Solution. Incubate 1 hour at 37°C.|4. Aspirate and wash 3 times.|5. Add 100ul prepared HRP Detection Working Solution. Incubate 30 minutes at 37°C.|6. Aspirate and wash 5 times.|7. Add 90ul Substrate. Incubate 15 minutes at 37°C.|8. Add 50ul Stop Solution. Read at 450nm immediately.